To more completely assess the fungal species to which people are exposed, a fungal Internal Transcribed Spacer (ITS) region gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States built environments are much more diverse than previously estimated using traditional methods of analysis. Fungal ITS sequencing data from samples collected from a variety of geographic regions within the United States including Atlanta (Georgia), Bennington (Vermont), Reno (Nevada) and Philadelphia (Pennsylvania) have been analyzed using Illumina miSeq high throughput sequencing or Sanger sequencing to characterize fungal communities in air and dust samples. The results of ITS region sequencing studies have also provided the first hazard identification data of fungi in occupational environments previously overlooked using traditional methods of assessment. For example, an analysis of area and personal air samples collected in occupational environments such as the emerging cannabis production industry has demonstrated a diversity of fungi primarily composed of species placed in the phylum Ascomycota including plant pathogenic species such as Botrytis cinerea, which causes grey mold. Other plant pathogens placed in the phylum Basidiomycota, including Wallemia sebi have been documented in another sampled production facility located in the US. An assessment of an office building in Bennington, Vermont has also shown the prevalence of overlooked fungal species such as, Ustilago syntherismae. Health assessments conducted via site visit have highlighted that workers can be exposed to microbial contaminants including fungal species that have been previously associated with occupational cases of hypersensitivity pneumonitis and allergic sensitization. As part of this series of studies, potential limitations associated with the sequencing technologies have been evaluated in recently completed work. While Sanger and Illumina sequencing resulted in similar fungal community profiles, the results of this study suggest that the ITS1 region provides the most reproducibility in identified fungal communities between sequencing platforms. Ongoing studies are examining the fungal communities present in the NYC Neighborhood Asthma and Allergy Study led by scientists located at Columbia University. Research performed by the collaborative study group has shown dust samples to contain fungal taxa including Aureobasidium pullulans, Penicillium glabrum, Wallemia sebi and Alternaria alternata. These prevalent fungal taxa varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. Additional studies suggest that multiple environmental factors including anthropogenic behavior modification, housing type, and neighborhood are important variables that influence fungal community profiles within middle-income homes in New York City. A. alternata measured in house dust was also associated with fractional exhaled nitric oxide, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation. Fungal diversity is also being compared to results obtained using qPCR and analysis of samples obtained from a variety of sources is ongoing. Work has also continued on the development of antibodies to recombinant fungal biomarker antigens. The utility of these antibodies is critical for the quantification of fungal biomarkers, particularly to those fungi that are being studied by the NTP. Epitope mapping experiments and optimization of immunoassays to identify fungal antigens are ongoing.